Optimal results were obtained by the addition of 3 mM magnesium oxalacetate, Fulvestrant clinical trial 5% v/v
DMSO and 8 μM primer concentration (Fig. 1). Lower primer concentrations produced less defined bands for primers OPL5 and RAPD5, and no amplification for primers P1 and P2 (data not shown). Similar observations were reported previously when typing Lactobacillus plantarum strains by RAPD-PCR in which the optimal primer concentration was also 8 μM (Johansson et al., 1995). As shown in Fig. 1, each primer generated distinct band patterns with amplicons ranging in size from approximately 500 bp to 12 kb. A total of 18 bands were observed for primer OPL5 (Fig. 1a), showing a greater discrimination among phages than the other primers that generated fewer (11–16) different bands (Fig. 1). With the exception of
S. epidermidis phages vB_SepiS-phiIPLA4, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA6, which had shown a closely related DNA restriction pattern, the RAPD-PCR band profiles were unique for each phage (Fig. 1). It is worth noting that L. lactis phage ΦC2 generated a small number of bands with all the primers assayed (Fig. 1, lane 7). INCB024360 cost Its lower genome size (22 163 bp) could explain this result (see Table 1). The genomic fingerprints resulting from the amplification of phage DNA samples performed on three separate days were compared to determine the RAPD-PCR reproducibility (Table 2). Each phage showed an identical band profile regardless of the assay date. Primers OLP5 and P2 provided high reproducibility values for genomic fingerprints and performed better than RAPD5 and P1. The low reproducibility of the later primers could be explained by the low number of amplification products obtained from phage ΦC2 with RAPD5 (see Fig. 1). Moreover, differences in the band
intensity on phage ΦH5 DNA may have accounted for the low reproducibility of P1 (data not Carnitine palmitoyltransferase II shown). No reproducible band intensities were likely due to nonspecific annealing between the primer and the DNA template as reported previously (Pérez et al., 1998). Phage suspensions were evaluated as source of DNA template to avoid the phage DNA purification step. Phage propagation in liquid and solid culture media yielded a titer of 107–108 and >109 PFU mL−1, respectively, for all selected phages. To discard amplification from bacterial DNA, noninfected host bacterial cultures were processed under the same conditions as the phage lysates and used as a template in RAPD-PCR reactions. No amplification from host DNA was observed under the assay conditions (data not shown). Moreover, genomic fingerprints obtained using both phage lysates (from liquid and solid medium propagation) as a template were apparently similar to each other and to those obtained using pure DNA as a template (see Fig. 2).